This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Targeted manipulation of retinoblastoma 1 protein, (pRb or RB1), suggested that Rb1 is required for normal hair cell (HC) cell cycle control, including full differentiation and mitotic quiescence (Mantela et al. 2005;Sage et al. 2005). However, global Rb1 deficient mice die before birth and exhibit an aberrant pattern of cell proliferation in the inner ear. Thus, a targeted excision of Rb1 in inner ear HCs is needed to avoid the deleterious global effects. Specific Rb1 deletion in the mouse HCs was recently shown to cause an aberrant proliferation at an early age, followed by a massive loss of HCs in the adults (Weber et al. 2005;Sage et al. 2006). This suggests that Rb1 imbalance in adult HCs leads to apoptosis, as expected from the deletion of either the Rb1 gene or its associated transcription factors: the E2Fs genes (Ikeda et al. 1996;Kastner et al. 1998;Giangrande et al. 2000;Hallstrom and Nevins 2003;Mantela et al. 2005). Nevertheless, data in the CNS and cardiac cells have shown the ability of some cell populations to re-enter the cell cycle and differentiate without apoptosis, suggesting that the impact of pRb ablation is likely cell-type dependent (Ferguson et al. 2002;MacPherson et al. 2003;Marino et al. 2003;Maclellan et al. 2005). The current results in Rb1 gene manipulation suggest that an alternative approach to study the potential of pRb manipulation for HC regeneration is needed. In order to accomplish this goal we have taken advantage of several of the novel molecular biology technologies which are described more in detail in our original proposal and briefly highlighted in the description of our specific aims (see bellow). Additionally, as per the COBRE internal council's suggestion, two new aims were added to this study. Initially, the potential of Rb1 manipulation in the inner ear hair cells to induce HCs regeneration will be assessed (S.A. 2). Our third aim will consist of using a transcriptomic approach to identify possible gene regulatory network for the differentiation of hair cells and supporting cells in the cochlea. Our ultimate goal is to manipulate postmitotic SC to re-enter the cell cycle and evaluate their potential to spontaneously differentiate into HCs.